This invention provides therapeutic compositions and combinations for the protection, treatment and repair of liver tissue. The invention relates to novel compositions and combinations comprising two or more compounds selected from the group consisting of S-adenosylmethionine, L-ergothioneine, and a substance selected from the group consisting of constituents of Milk thistle (Silybum marianum), silymarin and active components of silymarin, whether naturally, synthetically, or semi-synthetically derived, and to methods of preventing and treating liver disease and of repairing damaged liver tissue. The invention also provides a method of administering these compositions and combinations to humans or animals in need thereof.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention provides compositions and combinations for the protection, treatment and repair of liver tissue in humans and animals.

Additionally, the present invention provides such compositions and combinations that also produce a low level of side effects.

The present invention also provides a method of using the novel compositions and combinations of the present invention to protect, treat or repair liver tissue in humans or animals in need thereof.

The present invention provides novel compositions, combinations and methods for protecting, treating and repairing liver tissue. The compositions and combinations of the invention include two or more of the following compounds: S-adenosylmethionine, L-ergothioneine and a substance selected from the group consisting of a constituent of Milk thistle, silymarin and active components of silymarin, whether naturally, synthetically, or semi-synthetically derived.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the teachings of the present invention, disclosed herein are compositions, combinations and methods for the protection, treatment and repair of liver tissue. The invention relates to novel compositions and combinations comprising two or more compounds selected from the group consisting of S-adenosylmethionine, L-ergothioneine, and a substance selected from the group consisting of constituents of Milk thistle (Silybum marianum), silymarin and active components of silymarin, whether naturally, synthetically, or semi-synthetically derived, and to methods of preventing and treating liver disease and of repairing damaged liver tissue.

S-adenosylmethionine ("SAMe") (FIG. 1, see Original Patent) is a significant physiologic compound that is present throughout body tissue and that takes part in a number of biologic reactions as a methyl group donor or an enzymatic activator during the synthesis and metabolism of hormones, neurotransmitters, nucleic acids, phospholipids, and proteins. It is naturally formed in the body from ATP and methionine. SAMe is an extremely important reactant in many biochemical reactions including transmethylation, transsulfation, and synthesis of amines (FIG. 2, see Original Patent). Stramentinoli, G., Pharmacologic Aspects of S-Adenosylmethionine, American Journal of Medicine 83 (5A), 1987, pp. 35-42. In higher organisms, SAMe plays a significant role in transmethylation processes in more than 40 anabolic or catabolic reactions involving the transfer of the methyl group of SAMe to substrates such as nucleic acids, proteins and lipids, among others. The release of the methyl group from SAMe is also the start of a "transsulfuration" pathway that produces all endogenous sulfur compounds. After donating its methyl group, SAMe is converted into S-adenosylhomocysteine, which in turn is hydrolyzed to adenosine and homocysteine. The amino acid cysteine may then be produced from the homocysteine. Cysteine may exert a reducing effect by itself or as an active part of glutathione, which is a main cell antioxidant. Id. SAMe additionally has anti-oxidant effects via its derivatives (e.g., methylthioadenosine), which prevent oxidative damage to ells. Glutathione itself is a product of SAMe via the transmethylation and transsulfation pathways.

SAMe and its products, including glutathione, are of great importance in the prevention of liver damage. The changes produced by ethanol (EtOH) in the liver provide examples of injuries that can occur in the liver on the cellular level (FIG. 3, see Original Patent), and help explain the mechanism of action by which SAMe counteracts these injuries.

EtOH absorbed in the blood stream is metabolized in the liver by the enzyme alcohol dehydrogenase. This reaction releases excess nicotinamide-adenine-dinucleotide (NADH) which in turn shunts substrates (carbohydrates, lipids, and proteins) in the liver away from normal catabolic processes and towards lipid biosynthesis. As lipids accumulate in the liver cells in the form of large droplets, organelles are physically displaced and crowded, and this phenomenon decreases the cells' ability to function. Secondly, alcohol induces the P 450 system of cytochromes, and the microsomal ethanol oxidizing system ("MEOS") within liver cells, leading to augmented transformation of various compounds in the body (including, for example, chemicals from tobacco smoke) into toxic metabolites, and producing free radicals. Because alcohol consumption decreases glutathione pools, damage already produced by these free radicals is exacerbated. Alcohol and its metabolites (e.g., acetaldehyde) also interact with phospholipids and therefore have direct effects on hepatocellular membranes, decreasing their fluidity and affecting the function of organelles such as mitochondria and endoplasmic reticulum. Finally, acetaldehyde alters hepatocellular proteins, including the sodium/potassium pump, decreasing the ability of these proteins to function. The sodium/potassium pump is a membrane-bound protein that is responsible for maintaining the balance of sodium and potassium across the cell membrane of every cell in the body. Because many cell functions depend on the electrochemical gradient that results from this distribution of sodium and potassium, the sodium/potassium pump is essential to enable cells to perform. Liver cells are no exception. The alterations in proteins that alcohol and its metabolites induce also have the effect of making these proteins more `foreign` and thus more likely to induce autoimmune reactions. In short, alcohol damages the liver in a myriad of ways. FIG. 3; Lieber, C., Biochemical factors in alcoholic liver disease, Seminars in Liver Disease, 13 (2), 1993, pp. 136-53.

SAMe has a variety of beneficial effects in cells and protects hepatocytes from these injurious influences in a number of different ways. For example, SAMe has been shown to decrease lipid accumulation in rats chronically intoxicated with ethanol. This effect is not completely understood, but is partially explained by SAMe's ability to inhibit alcohol dehydrogenase. This single function of SAMe in itself prevents not only lipid accumulation but also much of the additional damage acetaldehyde causes to cellular membranes and proteins. Pascale, R., et al., Inhibition by ethanol of rat liver plasma membrane (Na+ K+)ATPase: protective effect of SAMe, L-methionine, and N-acetylcysteine, Toxicology and Applied Pharmacology, 97, 1989, pp. 216-29. Furthermore, because SAMe catalyses the transformation of phosphatidylethanolamine to phosphatidylcholine, it supports the normal fluidity of cell membranes, thereby supporting the structure and function of organelles including the plasma membrane, mitochondria and endoplasmic reticulum. This supportive effect avoids many of alcohol's damaging secondary effects. Bevi B., et al., Protection of rat fetal hepatocytes membranes from ethanol mediated cell injury and growth impairment, Hepatology 16, 1992, p. 109A.

SAMe also protects liver cells indirectly via its antioxidant products cysteine and glutathione, which help prevent damage by the excessive free radicals produced during alcohol intoxication. Pascale R., et al., The role of SAMe in the regulation of glutathione pool and acetaldehyde production in acute ethanol intoxication, Research Communications in Substances of Abuse, Vol. 5, No. 4, 1984, pp. 321-24.

Laboratory animal studies and in vitro experiments have verified these effects of SAMe on the inner, lipid layer of the plasma membrane. Champ, P. and Harvey, R., Biochemistry, 2.sup.nd ed., Lippincott, Philadelphia, 1994, pp. 266-7; Stramentinoli, G., Pharmacologic aspects of SAMe, American Journal of Medicine, Vol. 83 (5A) 1987, p. 35; Baldessarini, F., Neuropharmacology of S-Adenosyl Methionine, American Journal of Medicine 83 (5A), 1987, p. 95; Carney, M., Neuropharmacology of S-Adenosyl Methionine, Clinical Neuropharmacology 9 (3), 1986, p. 235; Janicak, P., S-Adenosylmethionine in Depression, Alabama Journal of Medical Sciences 25 (3), 1988, p. 306.

SAMe has been used to treat various disorders. In certain forms of liver disease, SAMe acts as an anticholestatic agent. Adachi, Y., et al., The Effects of S-adenosylmethionine on Intrahepatic Cholestasis, Japan Arch. Inter. Med., 33 (6), 1986, pp. 185-92. One mechanism by which SAMe exerts this effect is via its ability to maximize membrane fluidity, which is a crucial factor in the secretion of bile acids from hepatocytes. Id. Another mechanism is via the transsulfation pathway and the production of sulfates and taurine, which are important in mobilization of bile acids. Frezza, M., The use of SAMe in the treatment of cholestatic disorders, Drug Investigation, 4 (Suppl. 4), 1992, pp. 101-08. Low levels of SAMe are believed to play a role in increasing the risk of certain cancers. Feo F., et al., Early Stimulation of Polyamine Biosynthesis During Promotion by Phenobarbital of Diethylnitrosamine-induced Rat Liver Carcinogenesis. The Effects of Variations of the S-adenosyl-L-methionine Cellular Pool, Carcinogenesis, 6 (12), 1985, pp. 1713-20. The administration of SAMe has also been associated with a fall in the amount of early reversible nodules and the prevention of the development of late pre-neoplastic lesions and hepatocellular carcinomas. Garcea, R., et al., Variations of Ornithine Decarboxylase Activity and S-adenosyl-L-methionine and 5'-methylthioadenosine Contents During the Development of Diethylnitrosamine-induced Liver Hyperplastic Nodules and Hepatocellular Carcinoma, Carcinogenesis, 8 (5), 1987, pp. 653-58. SAMe is avaliable in many different salt forms as would be known by a person of ordinary skill in the art, any of which, or any combination of which, would be useful in the invention. SAMe and its salt forms may be natural, semisynthetic, bioengineered, synthetic or extracted, any of which, or any combination of which, would be useful in the invention.

L-ergothioneine (FIG. 4, see Original Patent) is a naturally occurring antioxidant that is very stable in the body. It is synthesized in fungi and microorganisms and present in both plants and animals. Animals are unable to synthesize L-ergothioneine and must obtain it from dietary sources. It is readily absorbed and is active in most mammalian tissues, concentrating especially in the liver, where it prevents certain types of free-radical-induced damage to cell membranes and organelles. For example, exogenous L-ergothioneine has been shown to prevent lipid peroxidation by toxic compounds in the liver tissue of rats. Akanmu, D., et al., The antioxidant action of ergothioneine, Arch. of Biochemistry and Biophysics, 288 (1), 1991, pp. 10-16; Kawano, H., et al., Studies on Ergothioneine: Inhibitory effect on lipid peroxide formation in mouse liver, Chem. Pharm. Bull., 31 (5), 1983, pp. 1662-87. In study comparing the inhibition of lipid peroxide ("LPO") formation by various compounds in mouse liver, L-ergothioneine both inhibited LPO formation and enhanced the decomposition of existing LPO (FIG. 5, see Original Patent). Id. L-ergothioneine additionally has been shown to inhibit the damaging effects caused by the oxidation of iron-containing compounds, such as hemoglobin and myoglobin. These molecules are important in the body as carriers of oxygen, but because they contain divalent iron, they can interact with hydrogen peroxide via the Fenton reaction to produce the even more damaging hydroxyl radical. This is the mechanism by which damage occurs during so-called reperfusion injury. Because L-ergothioneine acts as a reducing agent of the ferryl-myoglobin molecule, it can protect tissues from reperfusion injury. Hanlon, D., Interaction of ergothioneine with metal ions and metalloenzymes, J. Med. Chem., 14 (11), 1971, pp. 1084-87. Although L-ergothioneine does not directly scavenge superoxide anion or hydrogen peroxide, it contributes to the control of these free radicals by participating in the activation of superoxide dismutase and glutathione peroxidase. Its protective effects on cell membranes and other organelles are of benefit in acute and chronic toxicity as well as in infectious diseases, because common pathogenic biomechanisms are active in both of these processes. Ergothionine in any form would be useful in the invention, including natural, semisynthetic, bioengineered, synthetic, extracted and combinations thereof and including any other active forms, such as racemic mixtures (D & L forms). Because ergothioneine is available in nature, it is expected that daily microgram amounts will be effective as an antioxidant. Other antioxidants, such as selenium, are known to be effective as antioxidants at these very low levels.

Milk thistle (Silybum marianum) (FIG. 6, see Original Patent), which is also commonly known as Marian thistle, St. Mary's thistle, and Our Lady's thistle, is a native to the Mediterranean region, but has been naturalized in California and the eastern United States. This tall herb with prickly variegated leaves and milky sap has been used as a folk remedy for liver and biliary complaints for many years and recent research has supported such medicinal use. Foster, S., A Field Guide to Medicinal Plants, Houghton Mifflin Co, Boston, 1990, p. 198. Research over the past 20 years has documented that the plant contains a compound referred to as silymarin, which actually consists of various forms of hepatoprotectant flavonolignans. The principal components are silybin (which is also called silybinin); silychristin; and silydianin (which is also called silymonin); the 3-deoxy-derivatives of silychristin and silydianin; as well as isosilychristin; isosilybin (which is also known as isosilybinin) and its 3-deoxy derivative silandrin; the 3-deoxy compounds silyhermin A and B; 2,3 dehydrosilybin; and the trimers, quatramers and pentamers of silybin (which collectively are referred to as silybinomers). Other flavanolignans may be included as well. Isomers of silybin (or silybinin) are silybin A and B (or silybinin A and B). For purposes of this application, the term, "silybin" shall be used, but shall include silybinin. The structure of some of these are illustrated in: (FIG. 7, see Original Patent). Tyler, V., The Honest Herbal, Haworth Press, Inc., New York, 1993, pp. 209-10; Wichtl, M. (Grainger Bisset, N, trans.), Herbal Drugs and Phytopharmaceuticals, CRC Press, Boca Raton, 1994, pp. 121, 124, 125. These hepatoprotectant flavonolignans are referred to in this application as "active components of silymarin." The fruits (often erroneously referred to as the "seeds") of the plant, for example, contain approximately 3% flavonolignans on average. Laboratory trials in animals have shown that silymarins protect liver tissue against a variety of toxins including those of the deadly amanita mushrooms and carbon tetrachloride. Prophylactic effects were especially pronounced. Milk thistle is usually available as an extract that contains silymarin, but it is envisioned that any form or formulation of Milk thistle, e.g., extract, precipitate, or powdered form, which contains either silymarin or one or more active components of silymarin, would function in the present invention. In a preferred embodiment of the invention, the Milk thistle component or components may be "standardized," i.e., formulated so that a certain percentage or amount of a specific substance or of specific substances is or are present. As an example, the Milk thistle component of the invention (i.e. silymarin and the principal active components of silymarin, such as silybin, silydianin and silychristin) could be an extract. In that case, the extract can be standardized with respect to the percentage by weight of any or all of the silymarin constituents, particularly the silybin fractions present in the extract. For example, silymarin may be present in the extract in an amount from about 55% to about 85% by weight of the extract. In a more preferred embodiment, silymarin may be standardized so that it is present in an amount from about 67.5% to about 72.5% by weight of the extract. In another preferred embodiment the extract can be standardized to the amount of Silybinin A and Silybinin B, which may be present in a combined amount from about 20% to about 35% by weight, and most preferably about 28% by weight of the extract. In a still further preferred embodiment, Isosilybin A (also known as Isosilybinin A) and Isosilybin B (also known as Isosilybinin B) may be present in a combined amount from about 20% to about 35% by weight of the extract, as measured by HPLC (high pressure liquid chromatography). It may be possible to standardize the extract with respect to other flavonolignan fractions or isomers, such as dehydrosilybin, silydianin and silycristin, as well as their 3-deoxy derivatives. Each of these preferred embodiments may be present alone or in any combination. Recently, it has been shown that oral absorption of silymarin can be increased by combining the silimarin with phosphatidylcholine and this combination may also be used in the present invention.

Silymarin and the active components of silymarin have several mechanisms of action, including stimulation of nucleolar polymerase A. This stimulation in turn increases ribosomal activity leading to increased synthesis of cellular proteins, and an increased rate of hepatocellular repair. Conti, M., et al., Protective activity ofsilipide on liver damage in rodents, Japan J. Pharmacol., 60, 1992, pp. 315-21. Other protective mechanisms involve changes in the molecular structure of the hepatocellular membrane, which reduce binding and entry of toxins into the cell, and an antioxidant effect. Parish, R. & Doering, P., Treatment of Amanita mushroom poisoning: a review, Vet. Hum. Toxocol., 28 (4) 1986, pp. 318-22. It is expected that elements of the combinations of the present invention will work synergistically together because they have different, but complementary, mechanisms of action. Because liver diseases involve a complex interplay of numerous factors, the exact nature of which may remain obscure to the diagnosing clinician, there is a need for a composition that will address numerous mechanisms of liver damage. Treating the causative agent may not be--and in liver disease rarely is--possible. Addressing and preventing hepatic injuries on the cellular level therefore frequently will be the best treatment possible and almost as beneficial. The present invention combines antiinflammatory, anti-lipid, anti-necrotic, regenerating, and anti-fibrotic effects. All three ingredients that may be included in compositions of the present invention, S-adenosylmethionine, L-ergothioneine and a compound selected from the group consisting of Milk thistle, silymarin and active components of silymarin, have strong anti-inflammatory effects because of their antioxidant properties. Because different antioxidants have their primary effect on different free radicals, (for example, superoxide dismutase scavenges primarily superoxide anion), and because several types of free radicals are implicated in liver damage, supplying just one antioxidant would only address one subset of liver-damaging free radicals. It would also have a direct protective effect on protecting the hepatic cells when cells are stimulated by SAMe and or Silymarin to increase protein synthesis as this action of increased cell metabolism generates free radicals which can be neutralized by ergothionine.

Combining two of the three compounds will produce a beneficial effect in a number of liver diseases, and combining all three compounds will help treat or prevent an extremely broad range of such diseases. Thus, the compositions and combinations of the present invention will improve and maintain the health of liver tissue and normalize and improve the function of the liver in humans and animals. The combination will also allow beneficial effects to be achieved using lower doses than would otherwise be necessary. The use of lowered doses is both economically advantageous and reduces the risk of any potential side effects. Although the present ingredients are remarkably free of side effects, no compound is completely innocuous and giving the lowest effective dose is always sound medical policy.

The compositions and combinations of the present invention can be administered by a variety of routes including, but not limited to: orally, parentally, transdermally, sublingually, intravenously, intramuscularly, rectally and subcutaneously. Preferred daily doses for cach of the compounds are as follows. As would be apparent to a person of ordinary skill in the art, these dose ranges are approximations:

SAMe

Total dose range: about 5 mg--about 10 grams

Preferred small animal dose range: about 5 mg--about 1600 mg

Preferred human dose range: about 20 mg--about 5000 mg

Preferred large animal dose range: about 100 mg--about 10 grams

Alternatively, the daily per kilogram dose range of SAMe for all species is: about 2 mg/kg--about 100 mg/kg L-ergothioneine Total dose range: about 5 .mu.g--about 25 grams Preferred small animal dose range: about 5 .mu.g--about 5 grams Preferred human dose range: about 25 .mu.g--about 10 grams Preferred large animal dose range: about 100 .mu.g--about 25 grams Alternatively, the daily per kilogram dose range of L-ergothionine for all species is: about 2 .mu.g/kg--about 250 mg/kg Constituent of Milk thistle or silymarin, or active components of silymarin, (i.e., silybin, isosilybin, etc.) Total dose range: about 5 mg--about 10 grams Preferred small animal dose range: about 5 mg--about 1000 mg Preferred human dose range: about 100 mg--about 5 grams Preferred large animal dose range: about 250 mg--about 10 grams Alternatively, the daily per kilogram dose range of a consituent of Milk thistle, silymarin, or active components of silymarin for all species is: about 1 mg/kg--about 200 mg/kg

The daily doses recited above for all compounds may be given in a single dose or divided doses, to be administered, for example, twice-a-day, three-times a day or four-times-a-day. Therefore, the range for a single dose of the components of the invention is as follows: SAMe Total single dose range: about 1.25 mg--about 10 grams Preferred small animal single dose range: about 1.25 mg--about 1600 mg Preferred human single dose range: about 5 mg--about 5000 mg Preferred large animal single dose range: about 25 mg--about 10 grams Alternatively, the per kilogram single dose range of SAMe for all species is: about 0.5 mg/kg--about 100 mg/kg L-ergothioneine Total single dose range: about 1.25 .mu.g--about 25 grams Preferred small animal single dose range: about 1.25 .mu.g--about 5 grams Preferred human single dose range: about 6.25 .mu.g--about 10 grams Preferred large animal single dose range: about 25 .mu.g--about 25 grams Alternatively, the per kilogram single dose range for all species is: about 0.5 .mu.g/kg--about 250 mg/kg Constituent of Milk thistle (or silymarin, or active components of silymarin, ie., silybin, isosilybin, etc.) Total single dose range: about 1.25 mg--about 10 grams Preferred small animal single dose range: about 1.25 mg--about 1000 mg Preferred human single dose range: about 25 mg--about 5 grams Preferred large animal single dose range: about 62.5 mg--about 10 grams Alternatively, the per kilogram single dose range of a constituent of Milk thistle, silymarin, or active components of silymarin for all species is: about 0.25 mg/kg--about 200 mg/kg

Moreover, the dose may be administered in various combinations in which the components may be present in a single dosage form or in more than one dosage form. For example, the combinations of the present invention may be administered in a single daily dosage form in which all components are present, e.g., in a single capsule or tablet. The doses may also be administered in combinations of more than one dosage form in which each dosage form contains at least one component or in which two or more components are combined into a single dosage form. For example, a combination of SAMe and ergothioneine may be administered as a pill, capsule or tablet of SAMe and a separate pill, tablet or capsule of ergothioneine. A combination of ergothioneine, SAMe and silymarin may include each component in a separate dosage form, or two of the components in one dosage form, such as combined in the same capsule and the other component in a separate dosage form, or, as explained above, all three of the components in the same (i.e., a single) dosage form. These combinations may be provided in kits or blister packs, in which more than one dosage form of the various components are provided in the same package or container, for co-administration to a human or animal. For example, a tablet of SAMe and a capsule of silymarin can be placed in the same blister pack for co-administration. These combinations may be provided, for example, in kits, blister packs, packets or bottles shrink-wrapped together in which more than one dosage form of the various components are provided in the same dispensing unit for coadministration to a human or animal.
 

Claim 1 of 19 Claims

1. A composition comprising S-adenosylmethionine and a substance selected from the group consisting of silymarin and active components of silymarin.

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